Fluorescent protein sensors for measuring the pH of a biological sample

ABSTRACT

Disclosed are fluorescent protein sensors for measuring the pH of a sample, nucleic acids encoding them, and methods of use. The preferred fluorescent protein sensors are variants of the green fluorescent protein (GFP) from  Aequorea victoria . Also disclosed are compositions and methods for measuring the pH of a specific region of a cell, such as the mitochondrial matrix or the Golgi lumen.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.09/094,359, filed Jun. 9, 1998, issued on Oct. 31, 2000 as U.S. Pat. No.6,140,132, the disclosure of which is incorporated herein by referencein its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No. NS27177,awarded by the National Institutes of Health. The Government may haverights in this invention.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods formeasuring the pH of a sample and more particularly to fluorescentprotein sensors for measuring the pH of a biological sample.

BACKGROUND OF THE INVENTION

The pH within various cellular compartments is regulated to provide forthe optimal activity of many cellular processes. In the secretorypathway, posttranslational processing of secretory proteins, thecleavage of prohormones, and the retrieval of escaped luminalendoplasmic reticulum proteins are all pH-dependent.

Several-techniques have been described for measuring intracellular pH.Commonly used synthetic pH indicators can be localized to the cytosoland nucleus, but not selectively in organelles other than those in theendocytotic pathway. In addition, some cells are resistant to loadingwith cell-permeant dyes because of physical barriers such as the cellwall in bacteria, yeast, and plants, or the thickness of a tissuepreparation such as brain slices.

Several methods have been described for measuring pH in specific regionsof the cell. One technique uses microinjection of fluorescent indicatorsenclosed in liposomes. Once inside the cell, the liposomes fuse withvesicles in the trans-Golgi, and the pH of the intracellularcompartments is determined by observing the fluorescence of theindicator. This procedure can be laborious, and the fluorescence of theindicator can be diminished due to leakage of the fluorescent indicatorfrom the Golgi, or flux of the fluorescent indicator out of the Golgi aspart of the secretory traffic in the Golgi pathway. In addition, thefusion of the liposomes and components of the Golgi must take place at37° C.; however, this temperature facilitates leakage and flux of thefluorescent indicator from the Golgi.

A second method for measuring pH utilizes retrograde transport offluorescein-labeled verotoxin 1B, which stains the entire Golgi complexen route to the endoplasmic reticulum. This method can be used, however,only in cells bearing the receptor globotriaosyl ceramide on the plasmamembrane, and it may be limited by the residence time of the verotoxinin transit through the Golgi.

In a third method, intracellular pH has been measured using the chimericprotein CD25-TGN38, which cycles as between the trans-Golgi network andthe plasma membrane. At the plasma membrane, the CD25-motif bindsextra-cellular anti-CD25 antibodies conjugated with a pH-sensitivefluorophore. Measurement of fluorescence upon return of the boundcomplex to the Golgi can be used to measure the pH of the organelle.

SUMMARY OF THE INVENTION

The invention is based on the discovery that proteins derived from theAequorea victoria green fluorescence protein (GFP) show reversiblechanges in fluorescence over physiological pH ranges.

Accordingly, in one aspect, the invention provides a method fordetermining the pH of a sample by contacting the sample with anindicator including a first fluorescent protein moiety whose emissionintensity changes as the pH varies between 5 and 10, exciting theindicator, and the determining the intensity at a first wavelength. Theemission intensity of the first fluorescent protein moiety indicates thepH of the sample.

In another aspect, the invention provides a method for determining thepH of a region of a cell by introducing into the cell a polynucleotideencoding a polypeptide including a first fluorescent protein moietywhose emission intensity changes as the pH varies between 5 and 10,culturing the cell under conditions that permit expression of thepolynucleotide, and determining the intensity at a first wavelength. Theemission intensity of the first fluorescent protein moiety indicates thepH of the sample.

In a further aspect, the invention provides a functional engineeredfluorescent protein whose amino acid sequence is substantially identicalto the amino acid sequence of the 238 amino acid Aequorea Victoria greenfluorescence protein shown in FIG. 3 of U.S. Ser. No. 08/911,825, nowissued U.S. Pat. No. 6,054,321 (SEQ ID NO: 2), and whose emissionintensity changes as pH varies between 5 and 10.

In another aspect, the invention provides a polynucleotide encoding thefunctional engineered fluorescent protein.

The invention also includes a kit useful for the detection of pH in asample, e.g., a region of a cell. The kit includes a carrier meanscontaining one or more containers comprising a first containercontaining a polynucleotide encoding a polypeptide including a firstfluorescent protein moiety whose emission intensity changes as the pHvaries between 5 and 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting fluorescent protein sensors usedas indicators of intracellular pH.

FIGS. 2A and 2B are graphs showing absorbance as a function ofwavelength for the fluorescent protein pH sensor EYFP (SEQ ID NO: 6) atvarious wavelengths (FIG. 2A), and the pH dependency of fluorescence ofvarious GFP fluorescent protein sensors in vitro and in cells (FIG. 2B).The fluorescence intensity of purified recombinant GFP mutant protein(solid symbols) as a function of pH was measured in a microplatefluorometer. The fluorescence of the Golgi region of HeLa cellsexpressing proteins having the 81 N- terminal amino acids of the type IImembrane-anchored protein galactosyltransferase(GT:UDP-galactose-β,1,4-galactosyltransferase. EC 2.4.1.22) (“GT”) fusedto EYFP, or EGFP, i.e., GT-EYFP or ET-EGFP (open symbols) was determinedduring pH titration with the ionophores monensin/nigericin in high KCLsolutions.

FIGS. 3A and 3B are graphs showing ratiometric measurements of pH_(G) bycotransfecting HeLa cells with polynucleotides encoding GT-ECFP andGT-EYFP. FIG. 3A is a graph showing single-wave fluorescence intensitiesof GT-EYFP and GT-ECFP in the Golgi region of a HeLa cell. FIG. 3B is agraph showing the ratio of GT-EYFP/GT-ECFP fluorescence in the same cellas a function of time.

DETAILED DESCRIPTION

The invention provides genes encoding fluorescent sensor proteins, orfragments thereof, whose fluorescence is sensitive to changes in pH at arange between 5 and 10. The proteins of the invention are useful formeasuring the pH of a sample. The sample can be a biological sample andcan include an intracellular region of a cell, such as the lumen of themitochondria or golgi. The pH of a sample is determined by observing thefluorescence of the fluorescent sensor protein.

The fluorescent protein pH sensor have a broad applicability to cellsand organisms that are amenable to gene transfer. Problems associatedwith the use of other agents used to measure pH, e.g., problemsassociated with permeabilizing cells to ester-containing agents, leakageof agents, or hydrolysis of agents are avoided. With the fluorescentprotein pH sensors of the invention, no leakage occurs over the courseof a typical measurement, even when the measurement is made at 37° C.

Compositions and methods described herein also avoid the need to expressand purify large quantities of soluble recombinant protein, purify andlabel it in vitro, microinject it back into cells. An importantadvantage of the fluorescent protein pH sensors of the invention is theycan be delivered to cells in the form of polynucleotides encoding theprotein sensor fused to a targeting signal or signals. The targetingsignal directs the expression of the protein sensors to restricted celllocations. Thus, it is possible to measure the pH of a precisely definedcellular region or organelle.

POLYNUCLEOTIDES AND POLYPEPTIDES

In a first aspect, the invention provides a functional engineeredfluorescent protein whose amino acid sequence is substantially identicalto the 238 amino acid Aequorea Victoria green fluorescence protein shownin FIG. 3 of U.S. Ser. No. 08/911,825, now issued U.S. Pat. No.6,054,321 (SEQ ID NO:2). The term “fluorescent protein” refers to anyprotein capable of emitting light when excited with appropriateelectromagnetic radiation, and which has an amino acid sequence that iseither natural or engineered and is derived from the amino acid sequenceof Aequorea-related fluorescent protein. The term “fluorescent proteinpH sensor” refers to a fluorescent protein whose emitted light varieswith changes in pH from 5 to 10.

The invention also includes functional polypeptide fragments of afluorescent protein pH sensor. As used herein, the term “functionalpolypeptide fragment” refers to a polypeptide which possesses biologicalfunction or activity which is identified through a defined functionalassay and which is associated with a particular biologic, morphologic,or phenotypic alteration in the cell. The term “functional fragments ofa functional engineered fluorescent protein” refers to fragments of afunctional engineered protein that retain a function of the engineeredfluorescent protein, e.g., the ability to fluoresce in a pH-dependentmanner over the pH range 5 to 10. Biologically functional fragments canvary in size from a polypeptide fragment as small as an epitope to alarge polypeptide.

Minor modifications of the functional engineered fluorescent protein mayresult in proteins which have substantially equivalent activity ascompared to the unmodified counterpart polypeptide as described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polypeptides produced by thesemodifications are included herein as long as the pH-dependentfluorescence of the engineered protein still exists.

A functional engineered fluorescent protein includes amino acidsequences substantially the same as the sequence set forth in SEQ IDNO:2, and whose emission intensity changes as pH varies between 5 and10. In some embodiments the emission intensity of the functionalengineered fluorescent protein changes as pH varies between 5 and 8.5.

By “substantially identical” is meant a protein or polypeptide thatretains the activity of a functional engineered protein, or nucleic acidencoding the same, and which exhibits at least 80%, preferably 85%, morepreferably 90%, and most preferably 95% homology to a reference aminoacid or nucleic acid sequence. For polypeptides, the length ofcomparison sequences will generally be at least 16 amino If acids,preferably at least 20 amino acids, more preferably at least 25 aminoacids, and most preferably 35 amino acids. For nucleic acids, the lengthof comparison sequences will generally be at least 50 nucleotides,preferably at least 60 nucleotides, more preferably at least 75nucleotides, and most preferably 110 nucleotides.

By “substantially identical” is meant an amino acid sequence whichdiffers only by conservative amino acid substitutions, for example,substitution of one amino acid for another of the same class (e.g.,valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative substitutions, deletions, or insertions located atpositions of the amino acid sequence which do not destroy the functionof the protein (assayed, e.g., as described herein). Preferably, such asequence is at least 85%, more preferably 90%, more preferably 95%, morepreferably 98%, and most preferably 99% identical at the amino acidlevel to one of the sequences of EGFP (SEQ ID NO:4), EYFP (SEQ ID NO:6),ECFP (SEQ ID NO:8), or EGFP-V68L/Q69K (SEQ ID NO:10).

Homology is typically measured using sequence analysis software (e.g.,Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various substitutions, deletions,substitutions, and other modifications. Conservative substitutionstypically include substitutions within the following groups: glycinealanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine.

In some embodiments, the amino acid sequence of the protein includes oneof the following sets of substitutions in the amino acid sequence of theAequorea green fluorescent protein (SEQ ID NO:2): F64L/S65T/H231L,referred to herein as EGFP (SEQ ID NO:4); S65G/S72A/T203Y/H231L,referred to herein as EYFP (SEQ ID NO:6);S65G/V68L/Q69K/S72A/T203Y/H231L, referred to herein as EYFP-V68L/Q69K(SEQ ID NO:10); or K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L,referred to herein as ECFP (SEQ ID NO:8). The DNA sequences andcorresponding amino acid sequences of EGFP, EYFP, ECFP, andEYFP-V68L/Q69K are shown in Tables 1-8, respectively. The amino acidsare numbered with the amino acid following the iniating methionineassigned the ‘1’ position. Thus, F64L corresponds to a substitution ofleucine for phenylalanine in the 64th amino acid following the iniatingmethionine.

TABLE 1 EGFP Nucleic Acid Sequence (SEQ ID NO:3)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCGTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 2 EGFP Amino Acid Sequence (SEQ ID NO:4)MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

TABLE 3 EYFP Nucleic Acid Sequence (SEQ ID NO:5)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 4 EYFP Amino Acid Sequence (SEQ ID NO:6)MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

TABLE 5 ECFP Nucleic Acid Sequence (SEQ ID NO:7)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 6 ECFP Amino Acid Sequence (SEQ ID NO:8)MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

TABLE 7 EYFP-V68L/Q69K Nucleic Acid Sequaence (SEQ ID NO:9)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGAAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 8 EYFP-V68L/Q69K Amino Acid Sequence (SEQ ID NO:10)MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

In some embodiments, the protein or polypeptide is substantiallypurified. By “substantially pure protein or polypeptide” is meant anfunctional engineered fluorescent polypeptide which has been separatedfrom components which naturally accompany it. Typically, the protein orpolypeptide is substantially pure when it is at least 60% by weight,free from the proteins and naturally-occurring organic molecules withwhich it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, of the protein. A substantially pure protein may beobtained, for example, by extraction from a natural source (e.g., aplant cell); by expression of a recombinant nucleic acid encoding afunctional engineered fluorescent protein; or by chemically synthesizingthe protein. Purity can be measured by any appropriate method, e.g.,those described in column chromatography, polyacrylamide gelelectrophoresis, or by HPLC analysis.

A protein or polypeptide is substantially free of naturally associatedcomponents when it is separated from those contaminants which accompanyit in its natural state. Thus, a protein or polypeptide which ischemically synthesized or produced in a cellular system different fromthe cell from which it naturally originates will be substantially freefrom its naturally associated components. Accordingly, substantiallypure polypeptides include those derived from eukaryotic organisms butsynthesized in E. Coli or other prokaryotes.

The invention also provides polynucleotides encoding the functionalengineered fluorescent protein described herein. These polynucleotidesinclude DNA, cDNA, and RNA sequences which encode functional engineeredfluorescent proteins. It is understood that all polynucleotides encodingfunctional engineered fluorescent proteins are also included herein, aslong as they encode a protein or polypeptide whose fluorescent emissionintensity changes as pH varies between 5 and 10. Such polynucleotidesinclude naturally occurring, synthetic, and intentionally manipulatedpolynucleotides. For example, the polynucleotide may be subjected tosite-directed mutagenesis. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code.Therefore, all degenerate nucleotide sequences are included in theinvention as long as the amino acid sequence of the functionalengineered fluorescent protein or derivative is functionally unchanged.

Specifically disclosed herein is a polynucleotide sequence encoding afunctional engineered fluorescent protein that includes one of thefollowing sets of substitutions in the amino acid sequence of theAequorea green fluorescent protein (SEQ ID NO:2): S65G/S72A/T203Y/H231L,S65G/V68L/Q69K/S72A/T203Y/H231L, orK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L.

The term “polynucleotide” refers to a polymeric form of nucleotides ofat least 10 bases in length. The nucleotides can be ribonucleotides,deoxynucleotides, or modified forms of either type of nucleotide. Theterm includes single and double stranded forms of DNA. By “isolatedpolynucleotide” is meant a polynucleotide that is not immediatelycontiguous with both of the coding sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived. Theterm therefore includes, for example, a recombinant DNA which isincorporated into a vector, e.g., an expression vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences.

A “substantially identical” nucleic acid sequence codes for asubstantially identical amino acid sequence as defined above.

The functional engineered fluorescent protein can also include atargeting sequence to direct the fluorescent protein to particularcellular sites by fusion to appropriate organellar targeting signals orlocalized host proteins. A polynucleotide encoding a targeting sequencecan be ligated to the 5′ terminus of a polynucleotide encoding thefluorescence such that the targeting peptide is located at the aminoterminal end of the resulting fusion polynucleotide/polypeptide. Thetargeting sequence can be, e.g., a signal peptide. In the case ofeukaryotes, the signal peptide is believed to function to transport thefusion polypeptide across the endoplasmic reticulum. The secretoryprotein is then transported through the Golgi apparatus, into secretoryvesicles and into the extracellular space or, preferably, the externalenvironment. Signal peptides which can be utilized according to theinvention include pre-pro peptides which contain a proteolytic enzymerecognition site. Other signal peptides with similar properties topro-calcitonin described herein are known to those skilled in the art,or can be readily ascertained without undue experimentation.

The targeting sequence can also be a nuclear localization sequence, anendoplasmic reticulum localization sequence, a peroxisome localizationsequence, a mitochondrial localization sequence, or a localized protein.Targeting sequences can be targeting sequences which are described, forexample, in “Protein Targeting”, chapter 35 of Stryer, L., Biochemistry(4th ed.). W. H. Freeman, 1995. The localization sequence can also be alocalized protein. Some important targeting sequences include thosetargeting the nucleus (KKKRK, SEQ ID NO:15), mitochondrion (the 12 aminoterminal acids of the cytochrome c oxidase subunit IV gene or the aminoterminal sequence MLRTSSLFTRRVQPSLFRNILRLQST-, SEQ ID NO:16),endoplasmic reticulum (KDEL (SEQ ID NO:17) at C-terminus, assuming asignal sequence present at N-terminus), peroxisome (SKF at C-terminus),prenylation or insertion into plasma membrane (CaaX, CC, CXC, or CCXX atC-terminus), cytoplasmic side of plasma membrane (fusion to SNAP-25), orthe Golgi apparatus (fusion to the amino terminal 81 amino acids ofhuman type II membrane-anchored protein galactosyltransferase or fusionto furin).

Examples of targeting sequences linked to functional engineeredfluorescent proteins include GT-EYFP, GT-ECFP, GT-EGFP, andGT-EYFP-V68L/Q69K, which are targeted to the Golgi apparatus usingsequences from the GT protein; and EYFP-mito (SEQ ID NO:14) andEGFP-mito (SEQ ID NO:12), which are targeted to the mitochondrial matrixusing sequences from the amino terminal region of the cytochrome coxidase subunit IV gene. The EYFP, ECFP, EGFP, and EYFP-V68L/Q69K aminoacid sequences and corresponding polynucleotide

Polynucleotide sequences encoding EYFP-mito and ECFP-mito, along withtheir encoded amino acid sequences, are shown in Tables 9-12.

TABLE 9 ECFP-mito Nucleic Acid Sequence (SEQ ID NO:11)ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 10 ECFP-mito Amino Acid Sequence (SEQ ID NO:12)MLSLRQSIRFFKRSGIMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

TABLE 11 EYFP-mito Nucleic Acid Sequence (SEQ ID NO:13)ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 12 EYFP-mito Amino Acid Sequence (SEQ ID NO:14)MLSLRQSIRFFKRSGIMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

The fluorescent indicators can be produced as proteins fused to otherfluorescent indicators or targeting sequences by recombinant DNAtechnology. Recombinant production of fluorescent proteins involvesexpressing nucleic acids having sequences that encode the proteins.Nucleic acids encoding fluorescent proteins can be obtained by methodsknown in the art. For example, a nucleic acid encoding the protein canbe isolated by polymerase chain reaction of cDNA from A. Victoria usingprimers based on the DNA sequence of A. Victoria green fluorescentprotein. PCR methods are described in, for example, U.S. Pat. No.4,683,195; Mullis, et al. Cold Spring Harbor Symp. Quant. Biol. 51:263(1987), and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).Mutant versions of fluorescent proteins can be made by site-specificmutagenesis of other nucleic acids encoding fluorescent proteins, or byrandom mutagenesis caused by increasing the error rate of PCR of theoriginal polynucleotide with 0.1 mM MnCl₂ and unbalanced nucleotideconcentrations. See, e.g., U.S. patent application 08/337,915, filedNov. 10, 1994, now issued U.S. Pat. No. 5,625,048, or Internationalapplication PCT/US95/14692, filed Nov. 10, 1995, now published PCTApplication WO 96/23810.

The construction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques alsowell known in the art. Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,(Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., most recent Supplement).

Nucleic acids used to transfect cells with sequences coding forexpression of the polypeptide of interest generally will be in the formof an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used herein, “operatively linked” refers to ajuxtaposition wherein the components so described are in a relationshippermitting them to function in their intended manner. A control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with thecontrol sequences. “Control sequence” refers to polynucleotide sequenceswhich are necessary to effect the expression of coding and non-codingsequences to which they are ligated. Control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, components whose presence can influence expression, and canalso include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

As used herein, the term “nucleotide sequence coding for expression of”a polypeptide refers to a sequence that, upon transcription andtranslation of mRNA, produces the polypeptide. This can includesequences containing, e.g., introns. As used herein, the term“expression control sequences” refers to nucleic acid sequences thatregulate the expression of a nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalsfor introns, maintenance of the correct reading frame of that gene topermit proper translation of the mRNA, and stop codons.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the fluorescent indicator codingsequence and appropriate transcriptional/translational control signals.These methods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. (See, forexample, the techniques described in Maniatis, et al., Molecular CloningA Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₂ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransfected with DNA sequences encoding the fusion polypeptide of theinvention, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Techniques for the isolation and purification of polypeptides of theinvention expressed in prokaryotes or eukaryotes may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies or antigen.

A variety of host-expression vector systems may be utilized to expressfluorescent indicator coding sequence. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining a fluorescent indicator coding sequence; yeast transformedwith recombinant yeast expression vectors containing the fluorescentindicator coding sequence; plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobaccomosaic virus, TMV) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing a fluorescent indicator codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing a fluorescent indicator codingsequence; or animal cell systems infected with recombinant virusexpression vectors (e.g., retroviruses, adenovirus, vaccinia virus)containing a fluorescent indicator coding sequence, or transformedanimal cell systems engineered for stable expression.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, etc. may be used in the expression vector (see, e.g.,Bitter, et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used. When cloning in mammalian cell systems, promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the retrovirus long terminalrepeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of the insertedfluorescent indicator coding sequence.

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for thefluorescent indicator expressed. For example, when large quantities ofthe fluorescent indicator are to be produced, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Those which are engineered to contain acleavage site to aid in recovering fluorescent indicator are preferred.In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. & WileyInterscience, Ch. 13, 1988; Grant, et al., Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987,Acad. Press, N.Y., Vol. 153, pp.516-544, 1987; Glover, DNA Cloning, Vol.II, IRL Press, Wash., D.C., Ch. 3, 1986; and Bitter, Heterologous GeneExpression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.Press, N.Y., Vol. 152, pp. 673-684, 1987; and The Molecular Biology ofthe Yeast Saccharomyces, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II, 1982. A constitutive yeast promoter such as ADHor LEU2 or an inducible promoter such as GAL may be used (Cloning inYeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.11, A Practical Approach,Ed. D M Glover, IRL Press, Wash., D.C., 1986). Alternatively, vectorsmay be used which promote integration of foreign DNA sequences into theyeast chromosome.

In cases where plant expression vectors are used, the expression of afluorescent indicator coding sequence may be driven by any of a numberof promoters. For example, viral promoters such as the 35S RNA and 19SRNA promoters of CaMV (Brisson, et al., Nature 310:511-514, 1984), orthe coat protein promoter to TMV (Takamatsu, et al., EMBO J. 6:307-311,1987) may be used; alternatively, plant promoters such as the smallsubunit of RUBISCO (Coruzzi, et al., 1984, EMBO J. 3:1671-1680; Broglie,et al., Science 224:838-843, 1984); or heat shock promoters, e.g.,soybean hspl7.5-E or hspl7.3-B (Gurley, et al., Mol. Cell. Biol.6:559-565, 1986) may be used. These constructs can be introduced intoplant cells using Ti plasmids, Ri plasmids, plant virus vectors, directDNA transformation, microinjection, electroporation, etc. For reviews ofsuch techniques see, for example, Weissbach & Weissbach, Methods forPlant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463,1988; and Grierson & Corey, Plant Molecular Biology, 2d Ed., Blackie,London, Ch. 7-9, 1988.

An alternative expression system which could be used to expressfluorescent indicator is an insect system. In one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The fluorescent indicator coding sequence may becloned into non-essential regions (for example, the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example thepolyhedrin promoter). Successful insertion of the fluorescent indicatorcoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed, see Smith, et al., J. Viol. 46:584, 1983;Smith, U.S. Pat. No. 4,215,051.

Eukaryotic systems, and preferably mammalian expression systems, allowfor proper post-translational modifications of expressed mammalianproteins to occur. Eukaryotic cells which possess the cellular machineryfor proper processing of the primary transcript, glycosylation,phosphorylation, and, advantageously secretion of the gene productshould be used as host cells for the expression of fluorescentindicator. Such host cell lines may include but are not limited to CHO,VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38. Primary celllines, such as neonatal rat myocytes, can also be used.

Mammalian cell systems which utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, the fluorescent indicator coding sequencemay be ligated to an adenovirus transcription/translation controlcomplex, e.g., the late promoter and tripartite leader sequence. Thischimeric gene may then be inserted in the adenovirus genome by in vitroor in vivo recombination. Insertion in a non-essential region of theviral genome (e.g., region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing the fluorescent indicator ininfected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:3655-3659, 1984). Alternatively, the vaccinia virus 7.5K promoter may beused (e.g., see, Mackett, et al., Proc. Natl. Acad. Sci. USA, 79:7415-7419, 1982; Mackett, et al., J. Virol. 49: 857-864, 1984; Panicali,et al., Proc. Natl. Acad. Sci. USA 79: 4927-4931, 1982). Of particularinterest are vectors based on bovine papilloma virus which have theability to replicate as extrachromosomal elements (Sarver, et al., Mol.Cell. Biol. 1: 486, 1981). Shortly after entry of this DNA into mousecells, the plasmid replicates to about 100 to 200 copies per cell.Transcription of the inserted cDNA does not require integration of theplasmid into the host's chromosome, thereby yielding a high level ofexpression. These vectors can be used for stable expression by includinga selectable marker in the plasmid, such as the neo gene. Alternatively,the retroviral genome can be modified for use as a vector capable ofintroducing and directing the expression of the fluorescent indicatorgene in host cells (Cone & Mulligan, Proc. Natl. Acad. Sci. USA,81:6349-6353, 1984). High level expression may also be achieved usinginducible promoters, including, but not limited to, the metallothioneinIIA promoter and heat shock promoters.

The recombinant nucleic acid can be incorporated into an expressionvector including expression control sequences operatively linked to therecombinant nucleic acid. The expression vector can be adapted forfunction in prokaryotes or eukaryotes by inclusion of appropriatepromoters, replication sequences, markers, etc.

DNA sequences encoding the fluorescence indicator polypeptide of theinvention can be expressed in vitro or in vivo by DNA transfer into asuitable recombinant host cell. As used herein, “recombinant host cells”are cells in which a vector can be propagated and its DNA expressed. Theterm also includes any progeny of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. However,such progeny are included when the term “recombinant host cell” is used.Methods of stable transfer, in other words when the foreign DNA iscontinuously maintained in the host, are known in the art.

The expression vector can be transfected into a host cell for expressionof the recombinant nucleic acid. Recombinant host cells can be selectedfor high levels of expression in order to purify the fluorescentindicator fusion protein. E. coli is useful for this purpose.Alternatively, the host cell can be a prokaryotic or eukaryotic cellselected to study the activity of an enzyme produced by the cell. Inthis case, the linker peptide is selected to include an amino acidsequence recognized by the protease. The cell can be, e.g., a culturedcell or a cell taken in vivo from a transgenic animal.

TRANSGENIC ANIMALS

In another embodiment, the invention provides a transgenic non-humananimal that expresses a polynucleotide sequence which encodes afluorescent protein pH sensor.

The “non-human animals” of the invention comprise any non-human animalhaving a polynucleotide sequence which encodes a fluorescent indicator.Such non-human animals include vertebrates such as rodents, non-humanprimates, sheep, dog, cow, pig, amphibians, and reptiles. Preferrednon-human animals are selected from the rodent family including rat andmouse, most preferably mouse. The “transgenic non-human animals” of theinvention are produced by introducing “transgenes” into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 pl of DNA solution. The useof zygotes as a target for gene transfer has a major advantage in thatin most cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al., Proc. Natl. Acad. Sci. USA82:4438-4442, 1985). As a consequence, all cells of the transgenicnon-human animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene. Microinjection of zygotes is the preferred method forincorporating transgenes in practicing the invention.

The term “transgenic” is used to describe an animal which includesexogenous genetic material within all of its cells. A “transgenic”animal can be produced by cross-breeding two chimeric animals whichinclude exogenous genetic material within cells used in reproduction.Twenty-five percent of the resulting offspring will be transgenic, i.e.,animals which include the exogenous genetic material within all of theircells in both alleles. 50% of the resulting animals will include theexogenous genetic material within one allele and 25% will include noexogenous genetic material.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retro viral infection (Jaenisch, R., Proc. Natl. Acad. SciUSA 73:1260-1264, 1976). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida (Hogan, etal. (1986) in Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The viral vector systemused to introduce the transgene is typically a replication-defectiveretrovirus carrying the transgene (Jahner, et al., Proc. Natl. Acad.Sci. USA 82:6927-6931, 1985; Van der Putten, et al., Proc. Natl. Acad.Sci USA 82:6148-6152, 1985). Transfection is easily and efficientlyobtained by culturing the blastomeres on a monolayer of virus-producingcells (Van der Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (D. Jahner etal., Nature 298:623-628, 1982). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic nonhuman animal. Further, the founder maycontain various retro viral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retro viral infectionof the midgestation embryo (D. Jahner et al., supra). A third type oftarget cell for transgene introduction is the embryonal stem cell (ES).ES cells are obtained from pre-implantation embryos cultured in vitroand fused with embryos (M. J. Evans et al. Nature 292:154-156, 1981; M.O. Bradley et al., Nature 309: 255-258,1984; Gossler, et al., Proc.Natl. Acad. Sci USA 83: 9065-9069, 1986; and Robertson et al., Nature322:445-448, 1986). Transgenes can be efficiently introduced into the EScells by DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anonhuman animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. (Forreview see Jaenisch, R., Science 240: 1468-1474, 1988).

“Transformed” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant nucleic acid techniques, aheterologous polynucleotide. “Heterologous” refers to a polynucleotidesequence that either originates from another species or is modified fromeither its original form or the form primarily expressed in the cell.

“Transgene” means any piece of DNA which is inserted by artifice into acell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode which encodes the fluorescent indicator whichmay be expressed in a transgenic non-human animal. The term “transgenic”as used herein additionally includes any organism whose genome has beenaltered by in vitro manipulation of the early embryo or fertilized eggor by any transgenic technology to induce a specific gene knockout. Theterm “gene knockout” as used herein, refers to the targeted disruptionof a gene in vivo with complete loss of function that has been achievedby any transgenic technology familiar to those in the art. In oneembodiment, transgenic animals having gene knockouts are those in whichthe target gene has been rendered nonfunctional by an insertion targetedto the gene to be rendered non-functional by homologous recombination.As used herein, the term “transgenic” includes any transgenic technologyfamiliar to those in the art which can produce an organism carrying anintroduced transgene or one in which an endogenous gene has beenrendered non-functional or “knocked out.”

DETECTION OF pH USING FLUORESCENT INDICATOR PROTEINS

In another embodiment, the invention provides a method for determiningthe pH of a sample by contacting the sample with an indicator includinga first fluorescent protein moiety whose emission intensity changes aspH varies between pH 5 and 10, exciting the indicator, and thendetermining the intensity of light emitted by the first fluorescentprotein moiety at a first wavelength. The emission intensity of thefirst fluorescent protein moiety indicates the pH of the sample.

The fluorescent protein moiety can be a functional engineered proteinsubstantially identical to the amino acid sequence of Aequorea greenfluorescence protein (SEQ ID NO:2) Preferred green fluorescence proteinsinclude those having a functional engineered fluorescent protein thatincludes one of the following sets of substitutions in the amino acidsequence of the Aequorea green fluorescent protein (SEQ ID NO:2):S65G/S72A/T203Y/H231L, S65G/V68L/Q69K/S72A/T203Y/H231L, orK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L.

The sample in which pH is to be measured can be a biological sample,e.g., a biological tissue such as an extracellular matrix, blood orlymphatic tissue, or a cell. The method is particularly suitable formeasuring pH in a specific region of the cell, e.g., the cytosol, or anorganellar space such as the inner mitochondrial matrix, the lumen ofthe Golgi, cytosol, the endoplasmic reticulum, the chloroplast lumen,the lumen of lysosome, or the lumen of an endosome.

In some embodiments, the first fluorescent protein moiety is linked to atargeting sequence that directs the fluorescent protein to a desiredcellular compartment. Examples of targeting sequences include the aminoterminal 81 amino acids of human type II membrane-anchored proteingalactosyltransferase for directing the fluorescent indicator protein tothe Golgi and the amino terminal 12 amino acids of the presequence ofsubunit IV of cytochrome c oxidase for directing a fluorescent pHindicator protein to the mitochondrial matrix. The 12 amino acids of thepresequence of subunit IV of cytochrome c oxidase may be linked to thepH fluorescent indicator protein through a linker sequence, e.g.,Arg-Ser-Gly-Ile (SEQ ID NO:18).

In another embodiment, the invention provides a method of determiningthe pH of a region of a cell by introducing into the cell apolynucleotide encoding a polypeptide including an indicator having afirst fluorescent protein moiety whose emission intensity changes as pHvaries between 5 and 10, culturing the cell under conditions that permitexpression of the polynucleotide; exciting the indicator; anddetermining the intensity of the light emitted by the first proteinmoiety at a first wavelength. The emission intensity of the firstfluorescent protein moiety indicates the pH of the region of the cell inwhich the indicator is present.

The polynucleotide can be introduced using methods described above.Thus, the method can be used to measure intracellular pH in cellscultured in vitro, e.g., HeLa cells, or alternatively in vivo, e.g., incells of an animal carrying a transgene encoding a pH-dependentfluorescent indicator protein.

Fluorescence in the sample can be measured using a fluorometer. Ingeneral, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation which has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. If desired, a multi-axis translation stagecan be used to move a microtiter plate holding a plurality of samples inorder to position different wells to be exposed. The multi-axistranslation stage, temperature controller, auto-focusing feature, andelectronics associated with imaging and data collection can be managedby an appropriately programmed digital computer. The computer also cantransform the data collected during the assay into another format forpresentation.

Methods of performing assays on fluorescent materials are well known inthe art and are described in, e.g., Lakowicz, J. R., Principles ofFluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B.,Resonance energy transfer microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed.Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park:Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

The pH can be analyzed on cells in vivo, or from samples derived fromcells transfected with polynucleotides or proteins expressing the pHindicator proteins. Because fluorescent pH indicator proteins can beexpressed recombinantly inside a cell, the pH in an intracellularregion, e.g., an organelle, or an extracellular region of an organismcan be determined simply by determining changes in fluorescence.

Fluorescent protein pH sensors have may vary in their respective pK_(a),and the differences in pK_(a) can be used to select the most suitablefluorescent protein sensor most suitable for a particular application.In general, a sensor protein should be used whose pK_(a) is close to thepH of the sample to be measured. Preferably the pK_(a) is within 1.5 pHunit of the sample. More preferably the pK_(a) is within 1 pH unit, andstill more preferably the pK_(a) is within 0.5 pH unit of the sample.

Thus, a fluorescent protein pH sensor having a pK_(a) of about 7.1,e.g., the EYFP mutant described below, is preferred for determining thepH of cytosolic, Golgi, and mitochondrial matrix pH areas of a cell. Formore acidic organelles, a fluorescence sensor protein having a lowerpK_(a), e.g., a pK_(a) of about 6.1, is preferred.

To minimize artefactually low fluorescence measurements that occur dueto cell movement or focusing, the fluorescence of a the fluorescentprotein pH sensor can be compared to the fluorescence of a secondsensor, e.g., a second fluorescent protein pH sensor, that is alsopresent in the measured sample. The second fluorescent protein pH sensorshould have an emission spectra distinct from the first fluorescentprotein pH sensor so that the emission spectra of the two sensors can bedistinguished. Because experimental conditions such as focusing and cellmovement will affect fluorescence of the second sensor as well as thefirst sensor, comparing the relative fluorescence of the two sensorsallows for the normalization of fluorescence.

A convenient method of comparing the samples is to compute the ratio ofthe fluorescence of the first fluorescent protein pH sensor to that ofthe second fluorescent protein pH sensor.

KITS

The materials and components described for use in the methods of theinvention are ideally suited for the preparation of a kit. Such a kitmay comprise a carrier means being compartmentalized to receive one ormore container means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise apolynucleotide encoding a fluorescent protein pH sensor. A secondcontainer may further comprise fluorescent protein pH sensor. Theconstituents may be present in liquid or lyophilized form, as desired.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto limit the scope of the invention described in the claims.

EXAMPLES Example 1 Construction of Fluorescent Protein pH Sensors

Fluorescent protein pH sensors were constructed by engineeringsite-specific mutations in polynucleotides encoding forms of theAequorea victoria green fluorescent protein (GFP). The starting GFPvariant was the polynucleotide encoding the GFP variant EGFP (forenhanced green fluorescent protein). The EGFP variant had the amino acidsubstitutions F64L/S65T/H231L relative to the wild-type Aequoreavictoria GFP sequence.

The ECFP (enhanced cyan fluorescent protein) mutant was constructed byaltering the EGFP polynucleotide sequence so that it encoded a proteinhaving the amino acid substitutionsK26R/Y66W/N146I/M153T/V163A/N164H/H231, relative to the wild-type GFPamino acid sequence. A second variant, named EYFP (enhanced yellowfluorescent protein) was constructed by altering the EGFP polynucleotideto encode a protein having the amino acid substitutionsS65T/S72A/T203Y/H231L relative to the amino acid sequence of GFP. Athird variant, named EYFP-V68L/Q69K, was constructed by altering theEGFP polynucleotide to encode a protein having the amino acidsubstitutions S65G/V68L/Q69K/S72A/T203H231L relative to the amino acidsequence of GFP.

A HindIII site and Kozak consensus sequence (GCCACCATG) was introducedat the 5′ end of the polynucleotide encoding the GFP variants, and anEcoR1 site was added at the 3′ end of the gene of each indicator, andthe fragments were ultimately ligated into the HindIII/EcoR1 sites ofthe mammalian expression vector pcDNA3 (Invitrogen). EGFP and EYFPmutant proteins with no targeting signals were used as indicators of pHin the cytosol or nucleus indicators.

To construct fluorescent protein pH sensors to use as pH indicators inthe Golgi, polynucleotides encoding the 81 N-terminal amino acids of thetype II membrane-anchored protein galactosyltransferase(GT:UDP-galactose-β,1,4-galactosyltransferase. EC 2.4.1.22) ligated topolynucleotides encoding EGFP, ECFP, or EYFP. The polynucleotidesencoding the resulting proteins were named GT-EGFP, GT-ECFP, andGT-EYFP, respectively.

Mitochondrial matrix fluorescent protein pH sensors were constructed byattaching polynucleotides encoding 12 amino acids at the amino terminusto the presequence of subunit IV of cytochrome c oxidase (Hutl et al,EMBO J. 4:2061-68 (1985) to a polynucleotide encoding the amino acidsequence Arg-Sea-Gly-Ile (SEQ ID NO:18), which in turn was ligated topolynucleotides encoding ECFP or EYFP. These constructs were labeledECFP-mito or EYFP-mito.

The constructs used to examine intracellular pH are summarized in FIG.1.

Example 2 pH Titration of Fluorescent Sensor Proteins in Vitro

The pH sensitivity of the fluorescence of the proteins ECFP, EGFP, EYFP,GT-EGFP, and GT-EYFP was first examined.

Absorbance spectra were obtained in a Cary 3E spectrophotometer(Varian). For pH titration, a monochromator-equipped fluorometer (SpexIndustries, NJ) and a 96-well microplate fluorometer (CambridgeTechnology) were used. In the latter case the filters used forexcitation were 482±10 (460±18 for ECFP) and for emission were 532±14.Filters were named as the center wavelength ± the half-bandwidth, bothin nm. The solutions for cuvette titration contained 125 mM KCl, 20 mMNaCl, 0.5 mM CaCl₂, 0.5 mM MgCl₂, and 25 mM of one of the followingbuffers—acetate, Mes, Mops, Hepes, bicine, and Tris.

EYFP showed an acidification-dependent decrease in the absorbance peakat 514 nm and a concomitant increase in absorbance at 390 nm (FIG. 2A).The fluorescence emission (527-nm peak) and excitation spectra decreasedwith decreasing pH, but the fluorescence excitation spectrum showed nocompensating increase at 390 nm. Therefore, the species absorbing at 390nm was nonfluorescent. The apparent pK_(a) (pK'a) of EYFP was 7.1 with aHill coefficient (n) of 1.1 (FIG. 2B).

EGFP fluorescence also was quenched with decreasing pH. The pK'a of EGFPwas 6.15, and n was 0.7.

The change in fluorescence of ECFP (Tyr66→Trp in the chromophore) withpH was smaller than that of EGFP or EYFP (pK'a 6.4, n, 0.6) (FIG. 2B).The fluorescence change was reversible in the pH range 5-8.5 for allthree proteins, which covers the pH range of most subcellularcompartments. These results demonstrate that the GFP variants EGFP,EYFP, and ECFP can be used as fluorescent protein pH sensors.

Example 3 Measurements of pH in the Cytosol and Nucleus UsingFluorescent Protein pH Sensors

HeLa cells and AT-20 cells grown on glass coverslips were transientlylipo-transfected (Lipofectin™, GIBCO) with polynucleotide constructsencoding EYFP.

Cells were imaged between 2 and 4 days after transfection at 22° C. witha cooled charge-coupled device camera (Photometrices, Tucson, Ariz.) asdescribed in Miyawaki et al., Nature 388:882, (1997). The interferencefilters (Omega Optical and Chroma Technology, Brattleboro, Vt.) used forexcitation and emission were 440±10 and 480±15 for ECFP; 480±15 and535±22.5 for EGFP or EYFP. The dichroic mirrors were 455 DCLP for ECFPand 505 DCLP for EGFP or EYFP. Regions of interest were selectedmanually, and pixel intensities were spatially averaged after backgroundsubtraction. A binning of 2 was used to improve signal/noise andminimize photodamage and photoisomerization of EYFP. In the Cl-free bathsolution, Na-D gluconate and K-D gluoconate substituted for NaCl andHanks' balanced salt solution. High KCl buffer plus 5 μM each of theionophores nigericin (Fluka) and monensin (Calbiochem) was used for insitu titrations in living cells. Cells were loaded with cytosolic pHindicators by incubation with 3 μM carboxy- SNARF/AM or BCECF/AM(Molecular Probes) for 45 minutes, then washed for 30 minutes, all at22° C.

Fluorescence of HeLa cells transfected with the gene encoding EYFP wasdiffusely distributed in the cytosol and nucleus. This was expected fora protein of the size of GFP (27 kDa), which is small enough to passthrough nuclear pores.

The fluorescence observed with EYFP was reversible. Perfusion with NH₄Clcaused an increase in fluorescence (rise in pH), which reversed uponwashing out the NH₄Cl. Conversely, perfusion of lactate, which lowerspH, induced a decrease in fluorescence. The decrease in fluorescence wasalso reversible on wash-out.

Calibration of fluorescence intensity with pH in situ was accomplishedwith a mix of the alkali cation/H+ ionophores nigericin and monensin inbath solutions of defined pH and high K+. Fluorescence equilibratedwithin 1-4 minutes after each exchange of solution. These resultsdemonstrate that EYFP, when present intracellularly, can report pH inthe physiological range.

Example 4 Measurement of pH in the Mitochondrial Matrix UsingFluorescent Protein pH Sensors

To measure pH in the mitochondrial matrix using mutant GFP sensorproteins, HeLa cells and neonatal rat myocytes were transfected with thefluorescent protein pH sensor EYFP-mito. A Bio-Rad MRC-1000 confocalmicroscope was used for analysis of the targeted protein. Microscopyanalysis revealed that the transfected cells showed a fluorescencepattern indistinguishable from that of the conventional mitochondrialdye rhodamine 123.

In situ pH titration was performed with nigericin/monensin as describedin Example 3. Subsequent addition of the protonophore carbonylcyanident-chlorophenylhydrazone (CCCP) did not change the fluorescenceintensity of the cells. This demonstrates that the nigericn/monensintreatment effectively collapsed the pH gradient(ΔpH)in the mitochondria.

The estimated pHm was 7.98±0.07 in HeLa cells (n=17 cells, from sixexperiments). Similar pH values were obtained in a HeLa cell line stablyexpressing EYFP-mito. Resting pH did not change by superfusion of cellswith medium 10 mM glucose, which would provide cells with an oxidizablesubstrate, but 10 mM lactate plus 1 mM pyruvate caused an acidification,which reversed on washout. This can be accounted for by diffusion ofprotonated acid or by cotransport of pyruvate⁻/H⁺ through the innermitochondrial membrane. The protonophore CCCP rapidly induced anacidification of mitochondria to about pH 7.

Example 5 Measurement of pH in the Golgi Lumen Using Fluorescent ProteinpH Sensors

The type II membrane-anchored protein galactosyltransferase(GT:UDP-galactose-β,1,4-galactosyltransferase. EC 2.4.1.22) has beenused as a marker of the trans cisternae of the Golgi apparatus (Roth etal., J. Cell Biol. 93:223-29, (1982)). Accordingly, polynucleotideconstructs encoding portions of the GT protein fused to the mutant GFPproteins were constructed as described in Example 1 in order to use theGT sequence to target the fluorescent protein pH sensor to theendoplasmic reticulum.

The pH of the Golgi lumen was measured by transfecting HeLa or AT-20cells with the constructs GT-ECFP, GT-EGFP, or GT-EYFP. Brightjuxtanuclear fluorescence was observed, with little increase in diffusestaining above autofluorescnce in most cells.

The fluorescence pattern was examined further in double-labelingexperiments using rabbit polyclonal α-mannosidase II (α-manII) antibody.Double labeling fluorescence was performed as described by McCaffery etal., Methods Enzymol. 257:259-279 (1995). The α-manII antibody wasprepared as described in Velasco et al., J. Cell Biol. 122:39-51 (1993).In the double-staining experiments, it was observed that labeling of themedial trans-Golgi marker α-manII overlapped with GT-EYFP fluorescence.

α-manII was also fused with ECFP, and the pattern of fluorescenceobtained upon transfection of the gene was indistinguishable from thatof GT-EYFP by light microcopy.

To identify the subcellular localization of GT-EYFP at higherresolution, immunogold electron microscopy was performed on ultra-thincryosections by using antibodies against GFP. Immunogold labeling ofultra-thin sections was performed as described by McCaffery et al.,supra, using rabbit polyclonal anti-GFP antibody or a monoclonalanti-TGN38 antibody.

In double-labeling experiments, GT-EYFP was found in the medial andtrans Golgi, although endogenous GT is present in trans Golgi membranes.The difference in localization may occur as a result of overexpressionof the GT-EYFP protein.

When protein TGN38 was used as a trans-Golgi network (TGN) marker, itsimmunogold localization pattern was found to overlap with that ofGT-EYFP in the medial/trans-Golgi membranes. The localization datademonstrate that GT-EYFP labels the medial/trans Golgi. Thus, GT-EYFPcan be used to identify the pH of this organelle.

The pH titration of GT-EYFP fluorescence in the Golgi region of thecells after treatment with nigericin/monensin was in good agreement withthat of EYFP in vitro (see Example 2). Resting pH in HeLa cells was onaverage 6.58 (range 6.4-6.81, n=30 cells, 9 experiments). These resultsalso demonstrate that neither fusion with GT nor the composition of theGolgi lumen affects the pH sensitivity of EYFP. Thus, Golgi-targetedEYFP can be used as a local pH indicator.

The effect of various treatments on the pH of the Golgi was nextexamined using Golgi-targeted EYFP.

The pH gradient across the Golgi membrane is maintained by theelectrogenic ATP-dependent H⁺ pump (V-ATPase). The V-ATPase generates aΔpH (acidic inside) and Δψ (positive inside), which opposes further H⁺transport. The movement of counter-ions, Cl⁻ in (or K⁻ out), with H⁻uptake would shunt the Δψ, allowing a larger ΔpH to be generated. Thesemechanisms were investigated in intact single HeLa cells transfectedwith GT-EYFP.

The macrolide antibiotic bafilomycin Al has been shown to be a potentinhibitor of vacuolar type H⁺ ATPases (V type). In Hela cells expressingGT-EYFP, bafilomycin Al (0.2 μM) increased pH_(G) by about 0.6 units, topH 7.16 (range 7.02-7.37, n =12 cells. This suggests that the H⁺ pumpcompensates for a positive H+ efflux or leak. The initial rate of Golgialkalinization by bafilomycin Al was 0.52 pH units per minute (range0.3-0.77, n=12 cells), faster than that reported for other acidiccompartments such as macrophage phagosomes (0.09 pH/min). Similarresults regarding resting pHG and alkalinization by bafilomycin Al whereobtained when HeLa cells were transfected with GT-EGFP. Calibration ofGT-EGFP in situ also mirrored its in vitro titration (FIG. 1B). Thus,both EGFP and EYFP are suitable Golgi pH indicators.

Example 6 Measuring Intracellular pH With Two Fluorescent ProteinProtein Sensors

Quantitative measurements of fluorescence with nonratiometric indicatorscan suffer from artifacts as a result of cell movement or focusing. Tocorrect for these effects, the cyan-emitting mutant GT-ECFP wasco-transfected into cells along with GT-EYFP. ECFP has excitation andemission peaks that can be separated from those of EYFP by appropriatefilters. In addition, ECFP is less pH-sensitive than EYFP (see FIG. 2B).

FIG. 3A demonstrates that the fluorescence of ECFP changed less thanthat of EYFP during the course of the experiment. Although the ratio ofEYFP to ECFP emission varied between cells, probably reflecting adifferent concentration of GT-EYFP and GT-ECFP in the Golgi lumen, itchanged with pH as expected (FIG. 3B). Bafilomcin Al raised theGT-EYFP/GT-ECFP emission ratio, i.e, it raised pH_(G).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

18 1 716 DNA Aequorea victoria CDS (1)...(714) Green fluorescent protein1 atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 MetSer Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15gaa tta gat ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 GluLeu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggtgaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly GluGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 act actgga aaa cta cct gtt cca tgg cca aca ctt gtc act act ttc 192 Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggtgtt caa tgc ttt tca aga tac cca gat cat atg aaa cgg 240 Ser Tyr Gly ValGln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 cat gac tttttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe PheLys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt ttcaaa gat gac ggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe LysAsp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggtgat acc ctt gtt aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaagat gga aac att ctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tca cacaat gta tac atc atg gca gac aaa caa aag aat gga 480 Tyr Asn Ser His AsnVal Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gttaac ttc aaa att aga cac aac att gaa gat gga agc gtt 528 Ile Lys Val AsnPhe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gcagac cat tat caa caa aat act cca att ggc gat ggc cct 576 Gln Leu Ala AspHis Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt ttacca gac aac cat tac ctg tcc aca caa tct gcc ctt tcg 624 Val Leu Leu ProAsp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat cccaac gaa aag aga gac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro AsnGlu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 aca gct gctggg att aca cat ggc atg gat gaa cta tac aaa 714 Thr Ala Ala Gly Ile ThrHis Gly Met Asp Glu Leu Tyr Lys 225 230 235 ta 716 2 238 PRT Aequoreavictoria 2 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuVal 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val SerGly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys PheIle Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val ThrThr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His MetLys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr ArgAla Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met AlaAsp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp AsnHis Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn GluLys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala GlyIle Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 3 720 DNA Aequoreavictoria CDS (1)...(717) EGFP 3 atg gtg agc aag ggc gag gag ctg ttc accggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cacagg ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His ArgPhe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctgacc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu ThrLeu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc accctc gtg acc acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr LeuVal Thr Thr 50 55 60 ctg acc tac ggc gtg cag tgc ttc agc cgc tac ccc gaccac atg aag 240 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp HisMet Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tacgtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgcgcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg AlaGlu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctgaag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu LysGly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctggag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu GluTyr 130 135 140 aac tac aac agc cac aac gtc tat atc atg gcc gac aag cagaag aac 480 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln LysAsn 145 150 155 160 ggc atc aag gtg aac ttc aag atc cgc cac aac atc gaggac ggc agc 528 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu AspGly Ser 165 170 175 gtg cag ctc gcc gac cac tac cag cag aac acc ccc atcggc gac ggc 576 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile GlyAsp Gly 180 185 190 ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cagtcc gcc ctg 624 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln SerAla Leu 195 200 205 agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctgctg gag ttc 672 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu LeuGlu Phe 210 215 220 gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctgtac aag 717 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys225 230 235 taa 720 4 239 PRT Aequorea victoria 4 Met Val Ser Lys GlyGlu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu AspGly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu GlyAsp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr GlyVal Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His AspPhe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr IlePhe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val LysPhe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 IleAsp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly AspGly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln SerAla Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val LeuLeu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp GluLeu Tyr Lys 225 230 235 5 720 DNA Aequorea victoria CDS (1)...(717) EYFP5 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 MetVal Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15gtc gag ctg gac ggc gac gta aac ggc cac agg ttc agc gtg tcc ggc 96 ValGlu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 20 25 30 gagggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu GlyGlu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc accacc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 192 Cys Thr ThrGly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ttc ggc tacggc gtg cag tgc ttc gcc cgc tac ccc gac cac atg aag 240 Phe Gly Tyr GlyVal Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gacttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp PhePhe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttcttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe PheLys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gagggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu GlyAsp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aaggag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac aac agccac aac gtc tat atc atg gcc gac aag cag aag aac 480 Asn Tyr Asn Ser HisAsn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 ggc atc aaggtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 528 Gly Ile Lys ValAsn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 gtg cag ctcgcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 576 Val Gln Leu AlaAsp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 ccc gtg ctgctg ccc gac aac cac tac ctg agc tac cag tcc gcc ctg 624 Pro Val Leu LeuPro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205 agc aaa gacccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 672 Ser Lys Asp ProAsn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 gtg acc gccgcc ggg atc act ctc ggc atg gac gag ctg tac aag 717 Val Thr Ala Ala GlyIle Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 taa 720 6 239 PRTAequorea victoria 6 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val ValPro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Arg PheSer Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu ThrLeu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro ThrLeu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr ProAsp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro GluGly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn TyrLys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val AsnArg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn IleLeu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val TyrIle Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn PheLys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala AspHis Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu LeuPro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205 Ser Lys AspPro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val ThrAla Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 7 720DNA Aequorea victoria CDS (1)...(717) ECFP 7 atg gtg agc aag ggc gag gagctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu LeuPhe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gtaaac ggc cac agg ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val AsnGly His Arg Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tacggc aag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccctgg ccc acc ctc gtg acc acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro TrpPro Thr Leu Val Thr Thr 50 55 60 ctg acc tgg ggc gtg cag tgc ttc agc cgctac ccc gac cac atg aag 240 Leu Thr Trp Gly Val Gln Cys Phe Ser Arg TyrPro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg cccgaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro GluGly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tacaag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr LysThr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgcatc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg IleGlu Leu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg gggcac aag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Leu Glu Tyr 130 135 140 aac tac atc agc cac aac gtc tat atc acc gccgac aag cag aag aac 480 Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala AspLys Gln Lys Asn 145 150 155 160 ggc atc aag gcc cac ttc aag atc cgc cacaac atc gag gac ggc agc 528 Gly Ile Lys Ala His Phe Lys Ile Arg His AsnIle Glu Asp Gly Ser 165 170 175 gtg cag ctc gcc gac cac tac cag cag aacacc ccc atc ggc gac ggc 576 Val Gln Leu Ala Asp His Tyr Gln Gln Asn ThrPro Ile Gly Asp Gly 180 185 190 ccc gtg ctg ctg ccc gac aac cac tac ctgagc acc cag tcc gcc ctg 624 Pro Val Leu Leu Pro Asp Asn His Tyr Leu SerThr Gln Ser Ala Leu 195 200 205 agc aaa gac ccc aac gag aag cgc gat cacatg gtc ctg ctg gag ttc 672 Ser Lys Asp Pro Asn Glu Lys Arg Asp His MetVal Leu Leu Glu Phe 210 215 220 gtg acc gcc gcc ggg atc act ctc ggc atggac gag ctg tac aag 717 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp GluLeu Tyr Lys 225 230 235 taa 720 8 239 PRT Aequorea victoria 8 Met ValSer Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 ValGlu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 20 25 30 GluGly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 CysThr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 LeuThr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr130 135 140 Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln LysAsn 145 150 155 160 Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile GluAsp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr ProIle Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu SerThr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp HisMet Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu GlyMet Asp Glu Leu Tyr Lys 225 230 235 9 720 DNA Aequorea victoria CDS(1)...(717) EYFP-V68L/Q69K 9 atg gtg agc aag ggc gag gag ctg ttc acc ggggtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly ValVal Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aggttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Arg PheSer Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctg accctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr LeuLys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctcgtg acc acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu ValThr Thr 50 55 60 ttc ggc tac ggc ctg aag tgc ttc gcc cgc tac ccc gac cacatg aag 240 Phe Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His MetLys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtccag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val GlnGlu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gccgag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aagggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gagtac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr130 135 140 aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aagaac 480 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150 155 160 ggc atc aag gtg aac ttc aag atc cgc cac aac atc gag gacggc agc 528 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp GlySer 165 170 175 gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggcgac ggc 576 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly AspGly 180 185 190 ccc gtg ctg ctg ccc gac aac cac tac ctg agc tac cag tccgcc ctg 624 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser AlaLeu 195 200 205 agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctggag ttc 672 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu GluPhe 210 215 220 gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tacaag 717 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225230 235 taa 720 10 239 PRT Aequorea victoria 10 Met Val Ser Lys Gly GluGlu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp GlyAsp Val Asn Gly His Arg Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly AspAla Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly LysLeu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly LeuLys Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp PhePhe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile PhePhe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys PheGlu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile AspPhe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 AsnTyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser AlaLeu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu LeuGlu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu LeuTyr Lys 225 230 235 11 768 DNA Aequorea victoria CDS (1)...(765)ECFP-mito 11 atg ctg agc ctg cgc cag agc atc cgc ttc ttc aag cgc agc ggcatc 48 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Arg Ser Gly Ile 15 10 15 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg96 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 20 2530 gtc gag ctg gac ggc gac gta aac ggc cac agg ttc agc gtg tcc ggc 144Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 35 40 45gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 192 GluGly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 50 55 60 tgcacc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 240 Cys ThrThr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 65 70 75 80 ctgacc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg aag 288 Leu ThrTrp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 85 90 95 cag cacgac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 336 Gln His AspPhe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 100 105 110 cgc accatc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 384 Arg Thr IlePhe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 115 120 125 gtg aagttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 432 Val Lys PheGlu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 130 135 140 atc gacttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 480 Ile Asp PheLys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 145 150 155 160 aactac atc agc cac aac gtc tat atc acc gcc gac aag cag aag aac 528 Asn TyrIle Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 165 170 175 ggcatc aag gcc cac ttc aag atc cgc cac aac atc gag gac ggc agc 576 Gly IleLys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 180 185 190 gtgcag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 624 Val GlnLeu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 195 200 205 cccgtg ctg ctg ccc gac aac cac tac ctg agc acc cag tcc gcc ctg 672 Pro ValLeu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 210 215 220 agcaaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 720 Ser LysAsp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 225 230 235 240gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag 765 Val ThrAla Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 245 250 255 taa 76812 255 PRT Aequorea victoria 12 Met Leu Ser Leu Arg Gln Ser Ile Arg PhePhe Lys Arg Ser Gly Ile 1 5 10 15 Met Val Ser Lys Gly Glu Glu Leu PheThr Gly Val Val Pro Ile Leu 20 25 30 Val Glu Leu Asp Gly Asp Val Asn GlyHis Arg Phe Ser Val Ser Gly 35 40 45 Glu Gly Glu Gly Asp Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe Ile 50 55 60 Cys Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu Val Thr Thr 65 70 75 80 Leu Thr Trp Gly Val Gln Cys PheSer Arg Tyr Pro Asp His Met Lys 85 90 95 Gln His Asp Phe Phe Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu 100 105 110 Arg Thr Ile Phe Phe Lys AspAsp Gly Asn Tyr Lys Thr Arg Ala Glu 115 120 125 Val Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys Gly 130 135 140 Ile Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 145 150 155 160 Asn Tyr IleSer His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 165 170 175 Gly IleLys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 180 185 190 ValGln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 195 200 205Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 210 215220 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 225230 235 240 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys245 250 255 13 768 DNA Aequorea victoria CDS (1)...(765) EYFP-mito 13atg ctg agc ctg cgc cag agc atc cgc ttc ttc aag cgc agc ggc atc 48 MetLeu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Arg Ser Gly Ile 1 5 10 15atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 96 MetVal Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 20 25 30 gtcgag ctg gac ggc gac gta aac ggc cac agg ttc agc gtg tcc ggc 144 Val GluLeu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 35 40 45 gag ggcgag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 192 Glu Gly GluGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 50 55 60 tgc acc accggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 240 Cys Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 65 70 75 80 ttc ggc tacggc gtg cag tgc ttc gcc cgc tac ccc gac cac atg aag 288 Phe Gly Tyr GlyVal Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 85 90 95 cag cac gac ttcttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 336 Gln His Asp Phe PheLys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 100 105 110 cgc acc atc ttcttc aag gac gac ggc aac tac aag acc cgc gcc gag 384 Arg Thr Ile Phe PheLys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 115 120 125 gtg aag ttc gagggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 432 Val Lys Phe Glu GlyAsp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 130 135 140 atc gac ttc aaggag gac ggc aac atc ctg ggg cac aag ctg gag tac 480 Ile Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 145 150 155 160 aac tac aacagc cac aac gtc tat atc atg gcc gac aag cag aag aac 528 Asn Tyr Asn SerHis Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 165 170 175 ggc atc aaggtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 576 Gly Ile Lys ValAsn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 180 185 190 gtg cag ctcgcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 624 Val Gln Leu AlaAsp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 195 200 205 ccc gtg ctgctg ccc gac aac cac tac ctg agc tac cag tcc gcc ctg 672 Pro Val Leu LeuPro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 210 215 220 agc aaa gacccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 720 Ser Lys Asp ProAsn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 225 230 235 240 gtg accgcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag 765 Val Thr Ala AlaGly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 245 250 255 taa 768 14 255PRT Aequorea victoria 14 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe LysArg Ser Gly Ile 1 5 10 15 Met Val Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val Pro Ile Leu 20 25 30 Val Glu Leu Asp Gly Asp Val Asn Gly His ArgPhe Ser Val Ser Gly 35 40 45 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys LeuThr Leu Lys Phe Ile 50 55 60 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp ProThr Leu Val Thr Thr 65 70 75 80 Phe Gly Tyr Gly Val Gln Cys Phe Ala ArgTyr Pro Asp His Met Lys 85 90 95 Gln His Asp Phe Phe Lys Ser Ala Met ProGlu Gly Tyr Val Gln Glu 100 105 110 Arg Thr Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr Arg Ala Glu 115 120 125 Val Lys Phe Glu Gly Asp Thr LeuVal Asn Arg Ile Glu Leu Lys Gly 130 135 140 Ile Asp Phe Lys Glu Asp GlyAsn Ile Leu Gly His Lys Leu Glu Tyr 145 150 155 160 Asn Tyr Asn Ser HisAsn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 165 170 175 Gly Ile Lys ValAsn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 180 185 190 Val Gln LeuAla Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 195 200 205 Pro ValLeu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 210 215 220 SerLys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 225 230 235240 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 245 250255 15 5 PRT Homo sapiens 15 Lys Lys Lys Arg Lys 1 5 16 26 PRT Homosapiens 16 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val Gln Pro SerLeu 1 5 10 15 Phe Arg Asn Ile Leu Arg Leu Gln Ser Thr 20 25 17 4 PRTHomo sapiens 17 Lys Asp Glu Leu 1 18 4 PRT Aequorea victoria VARIANT(0)...(0) Linker sequence 18 Arg Ser Gly Ile 1

What is claimed is:
 1. A polynucleotide comprising a nucleotide sequenceencoding a functional engineered fluorescent protein whose emissionintensity changes as pH varies between 5 and 10, wherein said functionalengineered fluorescent protein is selected from the group consisting ofa) a protein comprising the substitutions S65G/S72A/T203Y/H231L withinthe amino acid sequence of Aequorea green fluorescent protein (SEQ IDNO:2), b) a protein comprising the substitutionsS65G/V68L/Q69K/S72A/T203Y/H231L within the amino acid sequence ofAequorea green fluorescent protein (SEQ ID NO:2), and c) a proteincomprising the substitutionsK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L within the amino acidsequence of Aequorea green fluorescent protein (SEQ ID NO:2), andwherein the amino acid sequence of said functional engineeredfluorescent protein is at least 95% homologous to the amino acidsequence of SEQ ID NO:2.
 2. An expression vector comprising at least oneexpression control sequence operatively linked to a polynucleotide ofclaim
 1. 3. A recombinant host cell comprising the expression vector ofclaim
 2. 4. The recombinant host cell of claim 3, wherein therecombinant host cell is a prokaryotic cell.
 5. The recombinant hostcell of claim 3, wherein the recombinant host cell is a eukaryotic cell.6. A method for determining the pH of a region of a cell comprising: a)introducing into the cell a polynucleotide encoding a protein comprisingan indicator having a fluorescent protein moiety, wherein thepolynucleotide encoding a protein comprising an indicator comprises thepolynucleotide of claim 1; b) culturing the cell under conditions thatpermit expression of the protein encoded by the polynucleotideintroduced into the cell; c) exciting the indicator; and d) determiningthe intensity of the light emitted by the fluorescent protein moiety ata wavelength, wherein the emission intensity of the fluorescent proteinmoiety indicates the pH of the region of the cell in which the indicatoris present.
 7. The method of claim 6, wherein said protein comprising anindicator further comprises a targeting sequence linked by a peptidebond to the indicator.
 8. The method of claim 7, wherein the targetingsequence comprises the amino terminal 81 amino acids of human type IImembrane-anchored protein galactosyltransferase.
 9. The method of claim7, wherein the targeting sequence comprises the amino terminal 12 aminoacids of the presequence of subunit IV of cytochrome c oxidase.
 10. Akit useful for the detection of the pH in a sample, the kit comprisingcarrier means containing one or more containers comprising a containercontaining a polynucleotide comprising a nucleotide sequence encoding afunctional engineered fluorescent protein whose emission intensitychanges as pH varies between 5 and 10, wherein said functionalengineered fluorescent protein is selected from the group consisting ofa) a protein comprising the substitutions S65G/S72A/T203Y/H231L withinthe amino acid sequence of Aequorea green fluorescent protein (SEQ IDNO:2), b) a protein comprising the substitutionsS65G/V68L/Q69K/S72A/T203Y/H231L within the amino acid sequence ofAequorea green fluorescent protein (SEQ ID NO:2), and c) a proteincomprising the substitutionsK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L within the amino acidsequence of Aequorea green fluorescent protein (SEQ ID NO:2), andwherein the amino acid sequence of said functional engineeredfluorescent protein is at least 95% homologous to the amino acidsequence of SEQ ID NO:2.
 11. A polynucleotide comprising a nucleotidesequence encoding a functional engineered fluorescent protein whoseemission intensity changes as pH varies between 5 and 10, wherein saidfunctional engineered fluorescent protein comprises a protein selectedfrom the group consisting of a) a protein comprising the substitutionsS65G/S72A/T203Y/H231L within the amino acid sequence of Aequorea greenfluorescent protein (SEQ ID NO:2), b) a protein comprising thesubstitutions S65G/V68L/Q69K/S72A/T203Y/H231L within the amino acidsequence of Aequorea green fluorescent protein (SEQ ID NO:2), and c) aprotein comprising the substitutionsK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L within the amino acidsequence of Aequorea green fluorescent protein (SEQ ID NO:2), andfurther comprises a conservative amino acid substitution in one or morepositions other than the amino acid positions specified in (a)-(c),wherein the amino acid sequence of said functional engineeredfluorescent protein is at least 95% homologous to the amino acidsequence of SEQ ID NO:2.
 12. A kit useful for the detection of the pH ina sample, the kit comprising carrier means containing one or morecontainers comprising a container containing a polynucleotide comprisinga nucleotide sequence encoding a functional engineered fluorescentprotein whose emission intensity changes as pH varies between 5 and 10,wherein said functional engineered fluorescent protein is selected fromthe group consisting of a) a protein comprising the substitutionsS65G/S72A/T203Y/H231L within the amino acid sequence of Aequorea greenfluorescent protein (SEQ ID NO:2), b) a protein comprising thesubstitutions S65G/V68L/Q69K/S72A/T203Y/H231L within the amino acidsequence of Aequorea green fluorescent protein (SEQ ID NO:2), and c) aprotein comprising the substitutionsK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L within the amino acidsequence of Aequorea green fluorescent protein (SEQ ID NO:2), andfurther comprises a conservative amino acid substitution in one or morepositions other than the amino acid positions specified in (a)-(c),wherein the amino acid sequence of said functional engineeredfluorescent protein is at least 95% homologous to the amino acidsequence of SEQ ID NO:2.